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Monday, June 18, 2012

The human machine: pistons and ratchets

In
my last post I talked about how we are all powered by tiny spinning
motors that work tirelessly to convert the chemical energy of our food into
electrical potential energy, and then back into chemical energy in the form of
the cellular energy currency, ATP . This week, I thought it would be
interesting to look at how that ATP gets used up in a process that will be very
familiar to you but that you probably know little about: muscle contraction.

As
I mentioned last time, ATP is used by just about every active process that
takes place in your cells. Most of these processes siphon only tiny amounts of
ATP from the ever-replenished pool that is available in your cells – as far as
they’re concerned there is an infinite amount of ATP available because they
could never use is all up on their own. For this reason, a lot of our cellular
machinery is, frankly, wasteful. The mechanisms that regulate DNA repair, movement of organelles within cells, and many other processes
consume ATP with gay abandon because their impact is so miniscule on the total
energy consumed by your body as a whole. There has been no need to evolve more
cost-efficient mechanisms, such as those employed by our single-celled relatives
for whom every ATP counts!

For
some components of the human machine, however, their impact on total ATP
consumption is far from insignificant. One often overlooked example is neural transmission, which is why the brain consumes around 20%
of your total calories on an average day, and why you may find yourself famished after a
tough exam even though you’ve just been sat down for three hours! Nonetheless,
the clear winner in the ATP-expenditure competition is muscle contraction. Your
muscles are responsible for using nearly 60% of your total calories on an average
day – potentially far more if you’re very active (or very muscular!). Motile
animals have to consume far more energy to survive than non-motile animals or
plants, and this is primarily down to their muscles. Fortunately, this is very
much worth it because being able to move gives you far broader options in terms
of finding food in the first place. Moreover, because of its huge significance
in terms of total energy consumption, the molecular basis of muscle contraction
has evolved to be a highly efficient affair. In fact, in terms of work achieved
per ATP used, muscle contraction is one of the most efficient processes in your
body – it’s simply the fact that it’s used on a huge scale that makes it such
an ATP-hungry mechanism.

The fine detail of mechanical beauty

Your musculature system is built on a very simple idea: individual muscles pull parts of the body in one direction, others in the opposite direction. Muscles never push, indeed they can’t push – they can only pull. The reason for this is found in the fine structure of the muscle tissue. All of your muscles are made up of a dense collection of
individual muscle fibres that pack together to form the entire muscle. These
fibres are made from special cells known as myocytes that fuse together
within the fibre to form one huge structure that acts, in effect, as one massive
cell. Each fibre is capable of independent contraction, in which it actively
shortens and thickens its whole structure. When every fibre in your muscle
contracts at once, the overall difference in length becomes enormous (on a
cellular level anyway) and so the familiar effect of muscle contraction is
achieved. Every movement you've ever made has been possible because of the
simultaneous cooperation of innumerable fibres working in unison. But how does
each fibre actually contract? Each one is further subdivided into cylindrical
structures known as myofibrils. These are the unit of contraction in
which the action happens. The rest of the fibre contains plenty of mitochondria to constantly pump out ATP for use in contraction, and a specialised
bag-like structure known as the sarcoplasmic reticulum that contains
heaps of calcium ions, the significance of which will be discussed later.

Your muscles are not as simple as they look!

As we move down to smaller and smaller scales, we learn that each myofibril also has an internal structure. It is at this scale that we finally meet the mechanical components responsible for contraction. Within each myofibril are a number of tiny fibres made up of repeating units of two proteins: actin and myosin. These proteins are organised into discrete units known as sarcomeres, in which actin (also known as the ‘thin’ fibre) is attached to the two ends of the sarcomere and extends into the centre, whilst myosin (the ‘thick’ fibre) is primarily attached to the centre and extends towards the ends. This generates a structure in which the two halves of the sarcomere are capable of moving relative to one another. When the fibre contracts, the two halves are pulled closer to one another, thereby shortening the whole sarcomere: hence the whole myofibril; the whole muscle fibre; and the whole muscle shortens. Pulling the two halves of the sarcomere together is the process that uses up so much of your ATP, and has evolved to be a highly efficient process.

The sarcomere. Every myofibril contains hundreds of sarcomeres laid end-to-end.

The process by which the sarcomere is shortened is a stroke of evolutionary genius that works with all of the elegance of any manmade machine. It all centres around what’s known as the ‘power stroke’ between actin and myosin. Actin itself doesn’t actually do any of the work, it simply provides a scaffold for myosin the latch onto in order to pull the two ends of the sarcomere together; myosin is the real workhorse. The structure of myosin contains a distinct head-like structure that protrudes out from the main myosin fibre and is capable of grabbing and moving actin. When you decide to contract a muscle, signals are sent from your brain to the nerves connecting to the muscle in question. These final nerves form a contact known as theneuromuscular junction, and when activated they release a neurotransmitter called acetylcholine onto the surface of the muscle fibres and the chain of events is now irreversibly set in motion that will cause contraction. Acetylcholine binding to receptors at the surface of the muscle fibres causes a cascade of signalling that sweeps through the whole fibre. The primary outcome of this is that the sarcoplasmic reticulum, which I mentioned earlier, disgorges all of its stored calcium into the surrounding myofibrils. Calcium plays a vital role in the regulation of muscle contraction because it binds to a protein called tropomyosin that winds its way around the actin fibre. In a resting muscle, tropomyosin sits in the clefts within the actin fibre that the myosin head is capable of binding to, and so actin and myosin are unable to interact. Calcium binds to tropomyosin and acts as a charge bridge (since calcium ions are positively charged) between two distinct parts of the protein. Pulling together these two sections of tropomyosin causes the whole shape of the protein to alter – the tropomyosin rotates relative to the actin filament and so exposes the myosin-binding site. This all happens without the need for ATP, although ATP is required to pump all of the calcium back into the sarcoplasmic reticulum at the end of contraction.

Now that myosin is free to bind actin, the power stroke can begin in earnest. To start with, the myosin head is bound to the two components that are used to make up ATP: ADP and Pi. In this state, its head is in an extended conformation and points away from the rest of the myosin fibre, and it is like this that it binds to actin. Once it is tightly latched on to the actin filament, the myosin head lets go of its ADP and Pi. This has a profound impact on myosin structure as the ADP and Pi are required to keep the head pointing away from the rest of myosin, without them the head swings backwards by 40˚, equating to a movement of around 6nm. Since it’s attached to actin, the actin also has to move that far to stay bound, and so the sarcomere is partially shortened. However, 6nm is not very much so the process must be repeated over and over again, gradually ratcheting the sarcomere shorter and shorter. In order for the cycle to restart we need our old friend ATP, which binds to the myosin head and stabilises a form in which it is able to release the actin. Nonetheless, the myosin head is still in its ‘contracted’ state and so must return to its original, extended conformation before the process can begin again. This is achieved by breaking down the newly-bound ATP into ADP and Pi, thus returning the system to its starting state. So, the energy that you use in muscle contraction is not actually directly used to make your muscle contract, but instead to allow the cycle to be reset. This is why a cramp in your leg is characterised by painful over-contraction of the muscle: you’ve over exerted yourself and so can’t produce enough ATP to allow efficient detachment of myosin from actin and thus can’t stop contracting. The same is true in the case of rigor mortis, as your muscles use up what little ATP is left in their cells and stiffen up never to be relaxed.

The overall effect of your muscles contracting is achieved
by billions of tiny power strokes taking place at unimaginable speed over and
over again until full contraction is reached. This is in turn powered by
countless ATP synthase motors spinning furiously to prevent a shortfall
in ATP, as mentioned in my previous post. We are, in a very real sense,
highly-tuned machines that have individual moving components working in unison
to accomplish set goals. Often when I’m running or cycling I will think about
the unimaginably enormous number of individual processes that have to constantly
rage in my muscles to allow me to keep going; but the same is true for any
movement at all – you evoke the same response just by moving your eyes along
this line of text.

What's left to learn?

The details that I have related to you above have been
slowly pieced together over the last hundred years or so in ever increasing
depth and understanding. We are now at a point where we understand the process
of muscle contraction and its regulation very well. However, many of the ways in which the structures that I have described above (sarcomeres, myofibrils, etc.) develop are still a mystery. Much headway has been made in recent years in our understanding of how the developmental processes behind these structures are regulated. This is often due to the investigation of a number or developmental disorders that highlight the role of specific gene and allow researchers to use that as a starting point. For example, our understanding of the role that theDMPK gene plays in muscle development has come primarily out of work looking into the genetic disorder myotonic dystrophy. Such investigations often have unexpected relevance to other fields since all biology is connected and we are unbelievably complex organisms. An example of this is the study of Emery-Dreifuss muscular dystrophy, a genetic disorder characterised by muscle wasting and cardiac problems. It was discovered that this is sometimes caused by mutations in genes encoding the lamin proteins. Lamins are responsible for the correct organisation of the nucleus within each cell, but this discovery revealed that correct muscle development is also highly dependent on them. Many of the proteins that are required for muscle development and contraction also have other roles (actin, in particular, acts as the internal skeleton for all cells), and much of the work that is currently ongoing is looking at how these different roles are reconciled with one another and how they are pushed into one job or the other.

All of this brings us closer to fully understanding our own complex construction, but also offers more immediate potential for the treatment of various disorders. For example, our understanding of what causes Duchenne muscular dystrophy has allowed the development of targeted gene therapies that may offer hope to sufferers by partially restoring normal function of their defective dystrophin genes. Similar trials are ongoing in several areas of neuromuscular developmental disorders, but none of it would be possible without a detailed biochemical understanding of how muscles work in the first place.

If you're at all interested, a highly interesting account of the evolution of muscle is given in Nick Lane's 'Life Ascending: The Ten Great Inventions of Evolution' along with, surprise, surprise, nine other cornerstones of life. It is definitely well worth a read!The next post in this series can be found here.

3 comments:

1) What changes in the story above when I choose to lift something heavy, compared to when I choose to lift something light. The physicist inside me tells me that I must be doing more work, and therefore must, presumably use more ATP up for the heavy thing; however it wasn't clear from the post how. Does it just require more iterations of the cycle to lift something heavy? That is, does one power stroke contract the muscle less if there is a resistive force acting on the muscle? Or is it some other way that the extra energy that must be being lost gets lost?

2) How did we learn the process you describe? I presume (and you wrote as much in your post) that it is built up over lots of different experiments, but what sort of experiments? I'm guessing it isn't as simple as pointing a microscope at a muscle and watching because (despite then pretty pictures) this stuff is happening at the chemical level so can't be seen. Is it done by making synthetic things based on guesses and seeing if they work correctly, or by removing certain chemicals from a lab-rat and seeing what stops working? (This question might be a bit too general... feel free to only be very vague in reply).

1. A very good question: every power stroke uses the same amount of energy and achieves the same absolute amount of sarcomere contraction, that doesn’t change depending on the force working against it (i.e. the weight being lifted). However, all of the events of the power stroke have to occur otherwise none of them does, which is to say that ADP and Pi can only be released if the myosin head can move, and the myosin head can only move if ADP and Pi can be released. If just one muscle fibre were trying to move something far too heavy, then the energy required would be too great and so the power stroke wouldn’t occur and the cycle would be stuck at the myosin-actin cross-bridge stage. In practice this means that if you lift something heavy you will use a lot more energy than if you try to lift something unliftable (like the floor or something) because you will go through lots of rounds of the cycle rather than just straining but never getting completing the power stroke. That said, if you did try to lift the floor then you would get tired because individually your muscle fibres would not all try to contract simultaneously but would rest and attempt to contract in pulses and so you would use some ATP, just less than if you were lifting a heavy yet manageable weight.

This leads somewhat into the answer to how heavy and light weights are moved differently. The simple answer is that you use more muscle fibres to lift heavy things than light things. If you do a bicep curl with a pencil then the nerve impulses to your bicep are relatively weak and so only a small proportion of all the fibres will contract. If you lift a 15kg weight then the pulse is much stronger and so more will contract. If you try to lift 30kg like this then it’s probably beyond the physical limit of what you bicep muscle alone can lift, and so to compensate you will engage other muscles (back, shoulders etc.) to bring extra force into the equation. At some point, though, you can’t argue with the physics and your muscles just simply aren’t capable of generating the necessary force to move whatever you’re trying to move – when you reach that point depends on how strong you are!

There is an extra complexity to this which is due to the fact that there are different types of muscle fibre. Broadly speaking, there are 2 types of fibre: slow and fast twitch. They vary primarily in the metabolic processes that generate ATP. I’m not going to go into detail here, but fast twitch muscles are capable of generating large amounts of ATP in a short space of time, primarily through a process called glycolysis, but can’t maintain it for long. Slow twitch muscles do the opposite: they can’t produce ATP at a really high rate, but can maintain a consistent output for a long time (primarily through mitochondrial metabolism). The ratio of one type of muscle to the next depends on genetic predisposition and the type of muscle: heart muscle, for example, is almost entirely slow, whereas the biceps are higher in fast twitch. If you’re lifting objects of different weight, you may engage different types of muscle fibre: slow twitch for light, fast for heavy, although it’s always a combination of both. Importantly, though, the mechanics of contraction and its physical limitations are identical in all muscle fibres.

2. As you suggested, this is a pretty broad question! The first detailed biochemical investigations looked at identifying the structure of fibres and muscles, primarily through microscopy. These showed the arrangement of the various filaments within the sarcomere and the organisation of the fibres in general. Then it was necessary to determine what the various filaments were made of, which was achieved through a number of biochemical means that can separate out different proteins and then identify their primary sequence (i.e. the combination and order of amino acids that make up the total protein). The techniques they used were crude by today’s standards but worked well enough mainly because they had absolutely tons of material to work with (muscle tissue is easy to come by – it’s just a steak!). Then they wanted to know roughly what was going on and what was needed to make it happen, which was done through a number of biophysical experiments where individual sarcomeres were isolated and then the different stages of the cycle identified in terms of their need for ATP and other components. These first showed that ATP is required for myosin release but not the power stroke, and that ADP/Pi release accompanies actin movement. The mechanical parameters (e.g. force generated, distance moved etc.) were often done by immobilising the myosin and actin and physically pulling on them with molecular tweezers – you can get the system to run in reverse if you add enough ADP/Pi and pull on the actin, which allows the quantification of the physics of the real process. These observations allowed a rough process to be outlined with the individual steps known in broad outcomes but not molecular detail. To get that kind of detail it was necessary to isolate actin and myosin and determine their structures in atomic resolution. This was done primarily through X-ray crystallographic approaches that show the position of every atom within the proteins (not easily done!). Each structure was just a snapshot of one state, but doing it in several states (ie. ATP-bound, ADP/Pi-bound, nothing bound) allowed the process to be slowly unpicked so that the cycle started to make sense in atomic detail. Once this was all worked out most work turned to looking at the subtler points of regulation, like the role of calcium or acetylcholine on contraction, which were done through standard molecular biology approaches, which I would love to go into in detail but I think you’d find it tedious pretty quickly!